Nucleotide sequence for creatinine deiminase and method of use

Abstract

The present invention relates to the discovery of novel genes encoding a polypeptide having creatinine deiminase activity and methods of use. Also disclosed is a kit using the novel sequences for determining the concentration of creatinine in a sample.

1. An isolated or purified nucleic acid coding for a polypeptide having creatinine deiminase activity, selected from the group consisting of:
 (a) the nucleic acid with a sequence as shown in SEQ ID NO: 1 or a fragment thereof coding for a polypeptide having creatinine deiminase activity; 
 (b) a nucleic acid having a sequence that is at least 95% identical to (a) and coding for a polypeptide having creatinine deiminase activity; and 
 (c) a nucleic acid having a degenerate form of the nucleic acid sequence according to (a) or (b). 
 
     
     
       2. The nucleic acid of  claim 1 , wherein the polypeptide having creatinine deiminase activity does not deaminate cytosine. 
     
     
       3. The nucleic acid of  claim 1 , wherein the nucleic acid is derived from  Tissierella creatinini.    
     
     
       4. A nucleic acid wherein the nucleic acid sequence is completely complementary to the full length sequence of the nucleic acid of  claim 1 . 
     
     
       5. The nucleic acid according to  claim 1 , wherein the nucleic acid comprises DNA or RNA. 
     
     
       6. A recombinant molecule, comprising a nucleic acid sequence according to  claim 1 . 
     
     
       7. The recombinant molecule according to  claim 6 , wherein the recombinant molecule is a vector or a plasmid. 
     
     
       8. The recombinant molecule according to  claim 7 , wherein the vector is a viral vector or a bacteriophage. 
     
     
       9. The recombinant molecule according to  claim 6 , further comprising an expression control sequence controlling the expression of the nucleic acid molecule. 
     
     
       10. The recombinant molecule according to  claim 9 , wherein the expression control sequence is homologous or heterologous to the nucleic acid molecule. 
     
     
       11. The recombinant molecule according to  claim 9 , wherein the expression control sequence comprises a promoter. 
     
     
       12. The recombinant molecule according to  claim 9 , wherein the expression of the recombinant molecule is controllable. 
     
     
       13. An isolated host cell comprising the molecule according to  claim 6 . 
     
     
       14. The host cell of  claim 13 , wherein the cell is selected from the group consisting of a prokaryotic cell, a yeast cell, an insect cell, a plant cell and a mammalian cell. 
     
     
       15. The host cell according to  claim 14 , wherein the prokaryotic cell is selected from the group consisting of  Escherichia coli  and  Bacillus subtilis.    
     
     
       16. The host cell of  claim 13 , wherein the nucleic acid molecule is expressed in the host cell to produce a polypeptide. 
     
     
       17. The host cell according to  claim 16 , wherein the polypeptide is secreted. 
     
     
       18. A method for preparing a polypeptide having a creatinine deiminase activity, said method comprising expressing in a host cell, a nucleic acid according to  claim 1  and isolating the polypeptide from the host cell. 
     
     
       19. The method according to  claim 18 , wherein isolating the polypeptide further comprises precipitating the polypeptide via ammonium sulphate and subjecting the polypeptide to chromatography using a sepharose containing column. 
     
     
       20. A kit for determining creatinine concentration in a sample, said kit comprising:
 (a) a nucleic acid sequence of  claim 1  or a host cell of  claim 13 ; and 
 (b) a reagent for determining the amount of ammonia formed in the sample.